Clh Report for 2,4-Dinitrophenol

CLH report

Proposal for Harmonised Classification and Labelling

Based on Regulation (EC) No 1272/2008 (CLP Regulation), Annex VI, Part 2

Substance Name: 2,4-Dinitrophenol

EC Number: 200-087-7 CAS Number: 51-28-5 Index Number: 609-041-00-4

Contact details for dossier submitter: Federal Institute for Occupational Safety and Health (BAuA) Federal Office for Chemicals Friedrich-Henkel-Weg 1-25 D-44149 Dortmund, Germany [email protected]

Version number: 1.0 Date: November 2017

CLH REPORT FOR 2,4-DINITROPHENOL

CONTENTS

Part A. 1 PROPOSAL FOR HARMONISED CLASSIFICATION AND LABELLING ...... 4 1.1 SUBSTANCE ...... 4 1.2 HARMONISED CLASSIFICATION AND LABELLING PROPOSAL ...... 4 1.3 PROPOSED HARMONISED CLASSIFICATION AND LABELLING BASED ON CLP REGULATION ...... 5 2 BACKGROUND TO THE CLH PROPOSAL ...... 7 2.1 HISTORY OF THE PREVIOUS CLASSIFICATION AND LABELLING ...... 7 2.2 SHORT SUMMARY OF THE SCIENTIFIC JUSTIFICATION FOR THE CLH PROPOSAL ...... 7 2.3 CURRENT HARMONISED CLASSIFICATION AND LABELLING ...... 7 2.3.1 Current classification and labelling in Annex VI, Table 3.1 in the CLP Regulation ...... 7 2.4 CURRENT SELF-CLASSIFICATION AND LABELLING ...... 7 2.4.1 Current self-classification and labelling based on the CLP Regulation criteria ...... 7 3 JUSTIFICATION THAT ACTION IS NEEDED AT COMMUNITY LEVEL ...... 8 Part B.

1 IDENTITY OF THE SUBSTANCE ...... 9 1.1 NAME AND OTHER IDENTIFIERS OF THE SUBSTANCE ...... 9 1.2 COMPOSITION OF THE SUBSTANCE ...... 10 1.2.1 Composition of test material ...... 10 1.3 PHYSICO-CHEMICAL PROPERTIES ...... 10 4 HUMAN HEALTH HAZARD ASSESSMENT ...... 13 4.1 TOXICOKINETICS (ABSORPTION, METABOLISM, DISTRIBUTION AND ELIMINATION) ...... 13 4.1.1 Non-human information ...... 13 4.1.2 Human information ...... 16 4.1.3 Summary and discussion on toxicokinetics ...... 19 4.2 ACUTE TOXICITY ...... 20 4.2.1 Non-human information ...... 20 4.2.1.1 Acute toxicity: oral ...... 20 4.2.1.2 Acute toxicity: inhalation ...... 23 4.2.1.3 Acute toxicity: dermal ...... 23 4.2.1.4 Acute toxicity: other routes ...... 24 4.2.2 Human information ...... 24 4.2.3 Summary and discussion of acute toxicity ...... 27 4.2.4 Comparison with criteria ...... 28 4.2.5 Conclusions on classification and labelling ...... 29 4.7 REPEATED DOSE TOXICITY ...... 29 4.7.1 Non-human information ...... 29 4.7.1.1 Repeated dose toxicity: oral ...... 29 4.7.1.2 Repeated dose toxicity: inhalation ...... 30 4.7.1.3 Repeated dose toxicity: dermal ...... 30 4.7.1.4 Repeated dose toxicity: other routes ...... 30 4.7.1.5 Human information ...... 30 4.7.1.6 Other relevant information ...... 30 4.7.1.7 Summary and discussion of repeated dose toxicity ...... 30 4.8 SPECIFIC TARGET ORGAN TOXICITY (CLP REGULATION) – REPEATED EXPOSURE (STOT RE) ...... 30 4.8.1 Summary and discussion of repeated dose toxicity findings relevant for classification as STOT RE according to CLP Regulation ...... 31 4.8.2 Comparison with criteria of repeated dose toxicity findings relevant for classification as STOT RE ...... 31 4.8.3 Conclusions on classification and labelling of repeated dose toxicity findings relevant for classification as STOT RE ...... 31 7 REFERENCES ...... 32

2 CLH REPORT FOR 2,4-DINITROPHENOL

Tables Table 1: Substance identity ...... 4 Table 2: The current Annex VI entry and the proposed harmonised classification ...... 4 Table 3: Proposed classification according to the CLP Regulation ...... 5 Table 4: Self-classification by the registrant (on March, 22nd, 2017) ...... 7 Table 5: Substance identity ...... 9 Table 6: Constituents (non-confidential information) ...... 10 Table 7: Impurities (non-confidential information) ...... 10 Table 8: Additives (non-confidential information) ...... 10 Table 9: Summary of physico - chemical properties ...... 10 Table 10: Summary table of relevant acute oral toxicity studies ...... 20 Table 11: Acute oral toxicity of dinitrophenol (Spencer et al., 1948) ...... 21 Table 12: Summary table of relevant acute dermal toxicity studies...... 23 Table 13: Fatal oral poisonings with information about doses applied ...... 26 Table 14: Fatal oral poisonings without information about doses applied ...... 27 Table 15: Summary table of relevant repeated dose toxicity studies ...... 29

3 CLH REPORT FOR 2,4-DINITROPHENOL Part A.

1 PROPOSAL FOR HARMONISED CLASSIFICATION AND LABELLING

1.1 Substance Table 1: Substance identity

Substance name: 2,4-dinitrophenol

EC number: 200-087-7

CAS number: 51-28-5

Annex VI Index number: 609-041-00-4

Degree of purity: Ca.85 %

Impurities: Considered confidential, please refer to the confidential annex.

1.2 Harmonised classification and labelling proposal

Table 2: The current Annex VI entry and the proposed harmonised classification

CLP Regulation SCL, M-Factor, ATE Current entry in Annex VI, CLP Acute Tox. 3*, H301, Regulation Acute Tox. 3*, H311, Acute Tox. 3*, H331, STOT RE 2*, H373**, Aquatic Acute 1, H400 Current proposal for consideration Acute Tox. 2, H300, ATE = oral by RAC 35 mg/kg bw Acute Tox. 3, H311, ATE = dermal STOT RE 2, H373 600 mg/kg bw Resulting harmonised classification Acute Tox. 2, H300, ATE = oral (future entry in Annex VI, CLP Regula- 35 mg/kg bw tion) Acute Tox. 3, H311, ATE = dermal Acute Tox. 3*, H331, 600 mg/kg bw STOT RE 2, H373, Aquatic Acute 1, H400

4 CLH REPORT FOR 2,4-DINITROPHENOL

1.3 Proposed harmonised classification and labelling based on CLP Regulation Table 3: Proposed classification according to the CLP Regulation CLP Hazard class Proposed clas- Proposed SCLs Current classifi- Reason for no Annex I sification and/or M-factors cation 1) classification 2) ref 2.1. Explosives Data lacking 2.2. Flammable gases Data lacking 2.3. Flammable aerosols Data lacking 2.4. Oxidising gases Data lacking 2.5. Gases under pressure Data lacking 2.6. Flammable liquids Data lacking 2.7. Flammable solids Data lacking 2.8. Self-reactive substances and Data lacking mixtures 2.9. Pyrophoric liquids Data lacking 2.10. Pyrophoric solids Data lacking 2.11. Self-heating substances and Data lacking mixtures 2.12. Substances and mixtures Data lacking which in contact with water emit flammable gases 2.13. Oxidising liquids Data lacking 2.14. Oxidising solids Data lacking 2.15. Organic peroxides Data lacking 2.16. Substance and mixtures cor- Data lacking rosive to metals 3.1. Acute Tox. 2 ATE = 35 mg/kg Acute Tox. 3* Acute toxicity - oral bw Acute Tox. 3 ATE = 600 mg/kg Acute Tox. 3* Acute toxicity - dermal bw Acute toxicity - inhalation Acute Tox. 3* 3.2. Not assessed in Skin corrosion / irritation this dossier 3.3. Serious eye damage / eye ir- Not assessed in ritation this dossier 3.4. Not assessed in Respiratory sensitisation this dossier 3.4. Not assessed in Skin sensitisation this dossier 3.5. Not assessed in Germ cell mutagenicity this dossier 3.6. Not assessed in Carcinogenicity this dossier 3.7. Not assessed in Reproductive toxicity this dossier

5 CLH REPORT FOR 2,4-DINITROPHENOL

CLP Hazard class Proposed clas- Proposed SCLs Current classifi- Reason for no Annex I sification and/or M-factors cation 1) classification 2) ref 3.8. Specific target organ toxicity Not assessed in –single exposure this dossier 3.9. Specific target organ toxicity STOT RE2 STOT RE2* – repeated exposure 3.10. Not assessed in Aspiration hazard this dossier 4.1. Hazardous to the aquatic en- Aquatic Acute 1 Not assessed in vironment this dossier 5.1. Not assessed in Hazardous to the ozone layer this dossier 1) Including specific concentration limits (SCLs) and M-factors 2) Data lacking, inconclusive, or conclusive but not sufficient for classification Labelling: Signal word: Danger Pictogram: GHS09 GHS08 GHS06 Hazard statements: H300 “Fatal if swallowed” H311 “Toxic in contact with skin” H331 “Toxic if inhaled” H373 “May cause damage to organs through prolonged or repeated Exposure” H400 “Very toxic to aquatic life” Proposed notes assigned to an entry: none

6 CLH REPORT FOR 2,4-DINITROPHENOL

2 BACKGROUND TO THE CLH PROPOSAL

2.1 History of the previous classification and labelling The current harmonised classification of dinitrophenol for acute toxicity and STOT RE 2 is the translation of the DSD classification with T; R23/24/25.

2.2 Short summary of the scientific justification for the CLH proposal It is one of the intentions of this proposal to remove the asterisks of the minimum classification for acute toxicity for the oral and the dermal route. Additionally, the asterisk for specific target organ toxicity – repeated exposure and the two asterisks for H373 shall be removed. This proposal is based on information available in the REACH-registration (accessed March 2017) and other information available in the scientific literature.

The proposed classification for acute oral toxicity with Acute Tox. 2; H300 is based on the LD50 range of 30 – 40 mg/kg bw in a key study on rats. This LD50 value fulfils the requirement for Acute Tox. 2, H300. Other studies and reports yielded LD50 values in similar ranges for other species such as mouse, rabbit and guinea pig. Several case reports from human poisonings suggested fatal doses of < 50 mg/kg bw. Only one study is available for acute dermal toxicity and this study confirmed the existing classification of Acute Tox. 3, H311. No information was available for acute inhalative toxicity and therefor no proposal for a change has been submitted. The classification for STOT RE 2 has been re-evaluated and confirmed on the basis of the CLP regulation.

2.3 Current harmonised classification and labelling

2.3.1 Current classification and labelling in Annex VI, Table 3.1 in the CLP Regulation Acute Tox. 3* (H301), Acute Tox. 3* (H311), Acute Tox. 3* (H331), STOT RE 2* (H373**), Aquatic Acute 1 (H400).

2.4 Current self-classification and labelling

2.4.1 Current self-classification and labelling based on the CLP Regulation criteria Table 4: Self-classification by the registrant (on March, 22nd, 2017) Harzard Class Statement Code # of notifiers Expl. 1.1 H201 4 Flam Sol. 1 H228 1 Acute Tox. 2 H300 5 Acute Tox. 3 H301 43 Acute Tox. 1 H310 1 Acute Tox. 3 H311 47

7 CLH REPORT FOR 2,4-DINITROPHENOL

Harzard Class Statement Code # of notifiers Acute Tox. 3 H331 47 STOT RE 2 H373 47 Aquatic Acute 1 H400 48 Skin Irrit. 2 H315 1 Muta. 2 H341 1 Repr. 2 H361 1 STOT SE 1 H370 1 STOT RE 1 H372 1 Aquatic Chronic 1 H410 1 Total number of notifiers: 48, Number of aggregated notifications: 7.

3 JUSTIFICATION THAT ACTION IS NEEDED AT COMMUNITY LEVEL 2,4-dinitrophenol is currently classified in Annex VI of the CLP regulation with a minimum classification for the acute toxicity hazard classes and STOT RE 2. Pursuant to section 4.1.1 and 4.2. of the guidance on the preparation of dossiers for harmonised classification and labelling, a refinement of such classifications warrants action at the EU level. Current classification of dinitrophenol for acute oral toxicity, Acute Tox. 3* is not adequate for this compound. Studies in several species and analysis of fatal human poisonings showed the necessity to correct the classification according to the LD50 values which are < 50 mg/kg bw. According to these values a classification of Acute Tox. 2, H300, seems to be appropriate for 2,4-dinitrophenol. During recent years several fatal poisonings with dinitrophenol happened in the United Kingdom (Bartlett et al. 2010, Siegmueller & Narasimhaiah 2010, Kamour et al. 2015) and Germany (BfR, 2015 (http://www.bfr.bund.de/cm/343/nahrungsergaenzungsmittel-die-dinitrophenol-dnp-enthalten- koennen-zu-schweren-vergiftungen-bis-hin-zu-todesfaellen-fuehren.pdf)). Dinitrophenol is adver- tised as diet aid (‘fat burner’) and distributed via internet based mail-order platforms. The effect is contributed to the suppression of the ATP (energy) production by uncoupling the oxidatative phos- phorylation of adenosine diphosphate in mitochondria. 2,4-DNP has a high acute toxicity causing fatal hyperthermia before death, other effects are increased basal metabolic rate, nausea, vomiting, sweating, dizziness, headache, loss of weight and cataracts. While its use in diet pills in the US were stopped in the 1930s, an increasing number of incidences of fatal overdoses including cases of intended suicide or intended weight/fat loss (of interest for people with body mass indices > 30 and bodybuilders) were observed after the year 2000. The classification as Acute Tox. 2, H300, would impose restrictions on placing on the market and distance sales in Germany for the substance on the basis of the German Prohibited Chemicals Act (Chemikalienverbotsverordnung).

8 CLH REPORT FOR 2,4-DINITROPHENOL

Part B.

SCIENTIFIC EVALUATION OF THE DATA

1 IDENTITY OF THE SUBSTANCE

1.1 Name and other identifiers of the substance Table 5: Substance identity

EC number: 200-087-7

EC name: 2,4-dinitrophenol

CAS number (EC inventory): 51-28-5

CAS number: 51-28-5

CAS name: Phenol, 2,4-dinitro-

IUPAC name: 2,4-dinitrophenol

CLP Annex VI Index number: 609-041-00-4

Molecular formula: C6H4N2O5

Molecular weight range: 184.11 g/mol

Structural formula:

9 CLH REPORT FOR 2,4-DINITROPHENOL

1.2 Composition of the substance

Table 6: Constituents (non-confidential information) Constituent Typical concentration Concentration range Remarks 2,4-dinitrophenol ca 85% (w/w) >80% - <90 % (w/w)

Table 7: Impurities (non-confidential information) Impurity Typical concentration Concentration range Remarks

Considered confidential, please refer to the confidential annex. Table 8: Additives (non-confidential information) Additive Function Typical concentration Concentration range Remarks none

1.2.1 Composition of test material

1.3 Physico-chemical properties Table 9: Summary of physico - chemical properties

Property Value Reference Comment (e.g. measured or estimated) State of the substance at Solid Visual inspection 20°C and 101,3 kPa (Manufacture as plate- lets, leaflets or crystal) Melting/freezing point 112-114 °C Secondary source that mentions: CRC Handbook of Chem- istry and Physics 88TH Edition 2007- 2008. CRC Press, Taylor & Francis, Boca Raton Secondary source1 Boiling point Not applicable, the O'Neil, M.J. (ed.). substance sublimes The Merck Index - An Encyclopedia of Chemicals, Drugs, and Biologicals. Whitehouse Station, NJ: Merck and Co., Inc., 2006., p. 556. Density 1.683 g/mL at 24 °C Secondary source that mentions: CRC Handbook of Chem- istry and Physics 88TH Edition 2007-

10 CLH REPORT FOR 2,4-DINITROPHENOL

Property Value Reference Comment (e.g. measured or estimated) 2008. CRC Press, Taylor & Francis, Boca Raton Secondary source1 Vapour pressure 1.49 x 10-5 mmHg at Secondary source 18 °C that mentions Mabey WR, Smith JH, Podoll RT, et al. 1981. Secondary source1 Surface tension - Substance is a solid. Water solubility 5.600 mg/L at 18 °C Secondary source that mentions: Har- vey DG., J Pharm

Pharmacol 11:462- 474., 1959; Second- ary source1 ______2790 mg/L at 20 °C secondary source EPISUITE 4.1 that mentions Schwarzenbach et al. 1988. Partition coefficient n- logPow 1.54 Secondary source octanol/water that mentions: Hansch C, Leo AJ., Claremont, CA: Pomona College. 1985; Secondary source1 Flash point - The study does not need to be conducted for explosives. Flammability - The study does not need to be conducted for explosives. Explosive properties Explosive or desensi- UN 0076 or UN No experimental data available tized explosive 1320, see United Nations Recommen- dations on the Transport of Dan- gerous Goods, Chap- ter 3.2, Rev. 19, 2015.1

1 Dinitrophenol in dry form has explosive properties. Because of its explosive properties, the compound is used in the form of a water paste. Regulations on the Transport of Dangerous Goods: In the United Nations Recommendations on the Transport of Dan- gerous Goods is an entry for Dinitrophenol (subsumed all isomers) which is assigned to be explosives and falls within Class 1 of those recommendations. The substance Dinitrophenol is specifically listed by name in the Dangerous Goods List in Chapter 3.2, and is assigned to two UN numbers: UN 0076: DINITROPHENOL, dry or wetted with less than 15% water, by mass; Class 1: Explosives - Division 1.1D: Substances and articles which have a mass explosion hazard UN 1320: DINITROPHENOL, WETTED with not less than 15% water, by mass; Class 4.1, UN packing group I: Solid desensitized explosives 11 CLH REPORT FOR 2,4-DINITROPHENOL

Property Value Reference Comment (e.g. measured or estimated) Self-ignition temperature - The study does not need to be conducted for explosives. Oxidising properties - The study does not need to be conducted for explosives. Solubility in organic solvents 2,4-dinitrophenol has Secondary source solubility at 15 °C that mentions: The (g/100 g solution): Merk Index, 1989; 15.55 in ethyl acetate; Secondary source2 35.90 in acetone; 5.39 in chloroform; 20.05 in pyridine; 0.423 in carbon tetra- chloride; 6.36 in toluene; soluble in alcohol and benzene. Dissociation constant pKa: 4.09 Secondary source that mentions: Pearce PJ, Simkins RJJ., Can J Chem 46:241-248., 1968 Secondary source: HSDB - Hazardous Substances Data Bank Viscosity - Substance is a solid.

The class of desensitized explosives comprises amongst others: (a) Solid desensitized explosives: explosive substances or mixtures which are wetted with water or alcohols or are dilut- ed with other substances, to form a homogeneous solid mixture to suppress their explosive properties. To classify desensitized explosives, data for the explosive potential and the corrected burning rate should be determined as described in Part V of the UN Recommendations on the Transport of Dangerous Goods, Manual of Tests and Crite- ria. However, the correct classification for “Desensitized explosives” in categories 1 to 4 could not be established because sufficient data are not available for the application of the classification criteria in GHS/CLP-Regulation. The correct classification shall be confirmed by testing. Furthermore, the substance has to comply with the requirements of Directive 2014/28/EU. 2 secondary source: M. Olivia Harris, M.A; James J. Corcoran, Ph.D. (peer rewied by Dr. martin Alexander; Dr.Leon Koller; Dr.Norman Trieff), U.S Department of Health and Human Services, Public Health Service Agency for Toxic Substances and Disease Registry 12 CLH REPORT FOR 2,4-DINITROPHENOL

4 HUMAN HEALTH HAZARD ASSESSMENT

4.1 Toxicokinetics (absorption, metabolism, distribution and elimination)

4.1.1 Non-human information Absorption Quotation from ATSDR (1995 and 2011): Oral absorption “Limited additional information is provided by animal studies. The half-time for absorption of 2,4- DNP following gavage administration of a single 22.5 mg/kg dose to mice was 0.5 hours (Robert and Hagardom 1983). A highly specific capillary gas chromatography (GC)-mass spectrometry (MS) technique was used to measure serum concentrations of 2,4-DNP at 1, 3, 6, 12, and 24 hours after dosing. The data were best represented by a two-compartment open model, and the analysis was performed accordingly. Fractional absorption was not determined. In an additional study employing the same analytical methods, 2,4-DNP (and its metabolites 2-amino-4-nitrophenol and 4- amino-2- nitrophenol) were monitored in plasma for 0.5, 1, 2, 4, 6, 9, 12, 24, 48, and 96 hours following a single gavage dose of 22.5 mg/kg (Robert and Hagardom 1985). Maximum values for the plasma concentration of 2,4-DNP were seen at 0.5 and 1.0 hours after dosing, giving additional evidence of rapid absorption.” “The time course of plasma concentrations of 2,4-DNP following oral administration to dogs (1 per dose) at 5, 12.5, 25, or 125 mg/kg indicated substantial absorption by the first sampling period, 0.5 hours (Kaiser 1964). Peak plasma levels were attained in 0.5-4 hours after dosing. In general, plasma levels tended to be higher in dogs that received higher doses. The 24-hour urine collections were analyzed for 2,4-DNP but not for metabolites; results were highly variable, raising the suspicion that collection may have been incomplete in some instances. Hence, the excretion data do not provide a reliable estimate of the fraction of the dose absorbed.” Inhalative exposure “No studies were located regarding absorption in animals after inhalation exposure to 2,4- DNP.” Dermal exposure “No studies were located regarding the rate or extent of absorption in animals after dermal exposure to 2,4-DNP. However, the death of 1 of 5 guinea pigs dermally exposed to 300 mg/kg (Spencer et al. 1948) suggests that dermal absorption occurred.”

Metabolism Quotation from ATSDR (1995 and 2011):

“Additional information regarding metabolism of 2,4-DNP is available from studies in animals. In a study from the older literature, 2,4-diaminophenol was identified in the urine of rabbits treated orally with 2,4-DNP, and was concluded to be a metabolite of 2,4-DNP (Ogino and Yasuku- ra 1957). The total dose given (probably to all the rabbits combined) was 30 grams (30,000 mg), and the total amount of 2,4-diaminophenol isolated from 10 liters of urine (presumably collected from all the dosed rabbits throughout the study) was 50 mg from 20 liters of urine. Hence, the yield

13 CLH REPORT FOR 2,4-DINITROPHENOL of this urinary metabolite was 0.17% of the dose. The metabolite was extracted from the urine, puri- fied, and identified by its properties, including melting point, nitrogen analysis, absorption curve, and various color reactions. Limitations of the study include the lack of adequate reporting of dose, route, and number of animals, relative lack of specificity in the identification methods available at the time, and the lack of experiments to quantitate losses of metabolite during the extraction and purification processes. Accordingly, only tentative conclusions regarding the identity of the metabolite as 2,4-diaminophenol and the percentage of the dose metabolized to this metabolite can be drawn from this study.” “2,4-DNP and two of its metabolites, 2-amino-4-nitrophenol and 4-amino-2-nitrophenol, were monitored in plasma of mice at 0.5, 1, 2, 4, 6, 9, 12, 24, 48, and 96 hours following a single gavage dose of 22.5 mg/kg using a highly specific capillary GC/MS technique (Robert and Hagar- dom 1985). The authors concluded that the amount of 2-amino-4-nitrophenol formed was 7.9 times the amount of 4-amino-2-nitrophenol, and that 50% of 2,4-DNP elimination involved direct conver- sion to these two compounds. Plasma concentrations of these two metabolites reached their highest levels within the first half hour after dosing, indicating rapid metabolism. The results demonstrate that 2-amino-4-nitrophenol is the major circulating metabolite of 2,4-DNP and that 4-amino-2- nitrophenol is also a significant circulating metabolite. The authors mentioned that they could not analyze for 2,4diaminophenol because of its reactivity with oxygen to form reactive quinones that could not be extracted and chromatographed by their method.“ “Analysis of urine for 2,4-DNP and its aminonitrophenol metabolites following a single subcutaneous injection of 20 mg/kg 2,4-DNP into rats revealed only parent compound and 2-amino- 4-nitrophenol (Parker 1952). 4-Amino-2-nitrophenol was not detected. The urine was collected over the 24-hour period following dosing.” “An in vitro study of 2,4-DNP metabolism using rat liver homogenates identified 2-amino- 4-nitrophenol and 4-amino-2-nitrophenol as metabolic products, with the 4-amino-2-nitrophenol present in greater abundance (Parker 1952). An additional ether-insoluble metabolite was tentative- ly identified as 2,4-diaminophenol. When 2-amino-6-nitrophenol or 4-amino-2-nitrophenol was incubated with rat liver homogenates, the 2-amino-4-nitrophenol was slowly metabolized to the ether-insoluble compound, while 4-amino-2-nitrophenol rapidly disappeared but with very little accumulation of the ether-insoluble compound. The reduction of the aminonitrophenols to the ether- insoluble compound appeared to be catalyzed by the same nitroreductase that reduces 2,4-DNP to the aminonitrophenols. A comparison of the activity of homogenates of various tissues in the rat and rabbit revealed that liver homogenate metabolized 2,4-DNP at a higher rate than did other tissue homogenates. In the rat, enzyme activities (mg 2,4-DNP metabolized per 100 grams wet weight of tissue) of other tissue homogenates relative to that of the liver homogenate (100%) were 60% in kidney, 59% in spleen, 47% in intrascapular fat, 29% in heart, 16% in muscle, and 3% in brain homogenates. In the rabbit, kidney homogenate activity was 41%, and heart homogenate activity was 3%, relative to liver homogenate activity. The rabbit spleen homogenate had no activity. The other rabbit tissues (intrascapular fat, muscle, and brain) were not analyzed. No activity was found in the blood of rats or rabbits.” “A more extensive investigation of the in vitro metabolism of 2,4-DNP by rat liver homogenates found that, under optimal pH and cofactor levels, 81% of the 2,4-DNP was metabolized. 2- Amino-4-nitrophenol accounted for 75%, 4-amino-2-nitrophenol for 23%, and 2,4-diaminophenol for ≈1% of the total amine metabolites produced (Eiseman et al. 1972). Even under suboptimal conditions, 2-amino-4nitrophenol was the predominant metabolite.” “The distribution of enzyme activity was analyzed in subcellular fractions: nucleic, mito- chondrial, microsomal, and cytosol (Eiseman et al. 1972). The maximum activity was found in the cytosol, which is the site of other nitroreductases, although nitroreductases can also be located in microsomes (Fouts and Brodie 1957; Juchau et al. 1970; Kamm and Gillette 1963; Kato et al. 1969; 14 CLH REPORT FOR 2,4-DINITROPHENOL

Parker 1952). The properties of nitroreductases have been extensively studied for the reduction of p-nitrobenzoic acid (Kato et al. 1969). Two separate enzyme systems are involved, one located in the cytosol, and the other in the microsomes. Both forms require the presence of reduced nicotina- mide adenine dinucleotides (NADH or NADPH) (Kato et al. 1969). The cytosolic reducing activity for 2,4-DNP required NADPH, since the activity in both the whole homogenate and in the cytosol was enhanced by adding glucose-6-phosphatase and NADP (Eiseman et al. 1972). The fact that the washed microsomal fraction contained no appreciable activity with 2,4-DNP could be due to the absence of soluble NADPH-generating enzymes, such as a glucose-6-phosphate dehydrogenase. Oxygen partially inhibited the formation of the aminonitrophenols. This inhibition is consistent with a reoxidation of cofactors FADH2 or NADPH in the presence of oxygen (Kamm and Gillette 1963). 14 Reduction of [ C]2,4-DNP to 2-amino-4-nitrophenol and 4-amino-2-nitrophenol by rat liver homogenates was not affected by the addition of p-nitrobenzoic acid, suggesting that different nitroreductases are involved (Eiseman et al. 1974). However, p-nitrophenol, o-nitrophenol, and 2,4- dinitro-6-sec-butylphenol inhibited the reduction of 2,4-DNP. The reduction was competitively inhibited by o-nitrophenol and noncompetitively inhibited by p-nitrophenol and 2,4-dinitro-6-sec- butylphenol. These results indicate separate metabolic pathways for 2,4-DNP and p-nitrobenzoic acid. The competitive inhibition by o-nitrophenol, however, suggests that 2,4-DNP and o- nitrophenol compete for the same active site on the nitroreductase, while the noncompetitive inhibition by the other two nitro compounds suggests binding at different sites on the enzyme.” “Limited information indicates that 2,4-DNP may also be conjugated to glucuronic acid or sulfate in the liver and then be excreted in the urine (NRC 1982).” “No studies were located regarding possible fecal metabolites of 2,4-DNP.”

Distribution Quotation from ATSDR (1995 and 2011): Oral exposure “Limited information is available regarding distribution in animals after oral exposure to 2,4-DNP. Pharmacokinetic analysis indicated that a two-compartment open model best characterized the disposition of 2,4-DNP in the serum, liver, and kidney of mice given a gavage dose of 22.5 mg/kg of 2,4-DNP (Robert and Hagardorn 1983). Serum and tissue levels of the parent compound were quantitated by a highly specific capillary GC-MS method at l-24 hours postdosing. Although concentrations of 2,4-DNP were much lower in liver and kidney than in serum, similar half-times for absorption (t1/2 = 0.50-0.62 hours) and for the fast (alpha) (t1/2 = 1.00-1.20 hours) phase of biphasic elimination in all 3 tissues were determined. However, terminal (beta) elimination phase from kidney was very slow (t1/2 = 76.2 hours) compared with the beta phase in liver (t1/2 = 8.7 hours) and in serum (t1/2 = 7.7 hours). The similar half-times for absorption and biphasic elimination in all three tissues (except terminal elimination phase in kidney) indicated that rapid exchange of 2,4-DNP occurred among these sites. The authors suggested that the apparent persistence of 2,4- DNP in the kidney could be related to tissue binding of the compound.” “The time course of plasma concentrations of 2,4-DNP following oral administration to dogs (1 per dose) at 5, 12.5, or 25 mg/kg gave no evidence of a trend towards higher plasma levels with continued daily dosing (Kaiser 1964). Hence, 2,4-DNP did not appear to accumulate.” Inhalation exposure “No studies were located regarding distribution in animals after inhalation exposure to 2,4-DNP”.

15 CLH REPORT FOR 2,4-DINITROPHENOL

Dermal exposure “No studies were located regarding distribution in animals after dermal exposure to 2,4-DNP.”

Elimination Quotation from ATSDR (1995 and 2011): Oral exposure “Pharmacokinetic analysis indicated that a two-compartment open model best characterized the disposition of 2,4-DNP in the serum, liver, and kidney of mice given a gavage dose of 22.5 mg/kg of 2,4-DNP (Robert and Hagardorn 1983). Serum and tissue levels of parent compound were quantitated by a highly specific capillary GC-MS method at l-24 hours postdosing. Half-times for the slow terminal elimination phases were 7.7 hours for serum, 8.7 hours for liver, and 76.2 hours for kidney. The authors suggested that the apparent persistence of 2,4-DNP in the kidney could be related to tissue binding of the compound.” “In an additional study employing the same analytical methods, 2,4-DNP and its metabolites, 2- amino-4-nitrophenol and 4-amino-2-nitrophenol, were monitored in plasma for 0.5-96 hours following a single gavage dose of 22.5 mg/kg in mice (Robert and Hagardorn 1985). Pharmacokinetic analysis indicated that two-compartment open models best characterized the disposition of 2,4-DNP and 2-amino-4-nitrophenol from plasma, whereas a three-compartment open model best characterized the disposition of 4-amino-2-nitrophenol from plasma. The elimination half-lives (t1/2) for the terminal phase were estimated at 10.3 hours for 2,4-DNP, 46.2 hours for 2-amino-4-nitrophenol, and 25.7 hours for 4-amino-2-nitrophenol.” “In dogs (1 per dose) that received 1, 12.5, or 25 mg/kg/day 2,4-DNP, 24-hour excretion of parent compound in the urine at 1, 3, and 6 days of treatment was erratic (Kaiser 1964), raising the suspicion that collection may have been incomplete in some instances. Greater amounts of 2,4-DNP were excreted after the first than after subsequent doses. Excretion of metabolites was not investigated.” “Species differences in elimination of 2,4-DNP do not appear to be large (less than two- fold) on the basis of a single limited study. The elimination rate constants for 2,4-DNP from blood of rats, rabbits, guinea pigs, and mice following unspecified single oral doses of 2,4-DNP were -1 0.062, 0.10, 0.12, and 0.098 hours , respectively (Lawford et al. 1954). “ Inhalation exposure “No studies were located regarding excretion in animals after inhalation exposure to 2,4- DNP.” Dermal exposure “No studies were located regarding excretion in animals after dermal exposure to 2,4-DNP.”

4.1.2 Human information Absorption Quotation from the ATSDR (1995 and 2011): Oral exposure 16 CLH REPORT FOR 2,4-DINITROPHENOL

“The data regarding absorption in humans after oral exposure are limited. Evidence of substantial 2-4-DNP absorption was obtained from the case of an 80-kg man who ingested two 4.5-g doses of the sodium salt of 2,4-DNP (each equivalent to 46 mg 2,4-DNP/kg) 1 week apart and died 11 hours after the second dose (Tainter and Wood 1934). Analysis of a blood sample for 2,4- dinitrophenol and estimation of the total body burden, assuming the drug was evenly distributed between blood and tissues, gave a body burden of ≈2.72 grams 2,4-DNP, which corresponds to 3.31 grams of the sodium salt of 2,4-dinitrophenol at the time of death. Since some of the drug would have been metabolized, and some excretion of parent compound and metabolites probably would have occurred during the interval between ingestion and death, this value is not inconsistent with complete absorption of the second dose. 2,4-DNP and 2-amino-4-nitrophenol were detected in the urine of a woman who had taken sodium 2,4-DNP at 3.5 mg/kg/day 2,4-DNP for 20 days (Davidson and Shapiro 1934), indicating that absorption had occurred. Quantitative data were not reported. Indirect evidence of rapid absorption is provided by the maximal increases in basal metabolic rate that occurred within 1 hour of ingestion of 2-5 mg/kg 2,4-DNP from 2,4-DNP (Cutting et al. 1933) or sodium 2,4-DNP (Dunlop 1934) by patients in clinical studies.” Inhalative exposure “No quantitative data were located regarding absorption in humans after inhalation exposure to DNPs. A metabolite of 2,4-DNP, 2-amino-4-nitrophenol, was commonly detected by the Derrien test in the urine of workmen (women were generally not employed in dangerous processes) exposed via inhalation to vapor and airborne dust of 2,4-DNP and by direct contact of the skin with the solid chemical in the munitions industry in France (Perkins 1919). Exposure may have occurred by the dermal and possibly oral routes, as well as by inhalation. In addition, examination of the blood, unspecified organs, and urine of workmen in this industry who died from exposure to 2,4-DNP revealed the presence of 2,4-DNP and its metabolites; quantitative data were not provided (Perkins 1919). Despite its limitations, the study provides some evidence of absorption from inhalation exposure.” “In a case of fatal occupational 2,4-DNP poisoning from exposure to mists and airborne dust of 2,4- DNP in the U.S. chemical industry, the urine contained 2.08 g/L of 2,4-DNP and 50 mg/L of 2- amino-4-nitrophenol (Gisclard and Woodward 1946). Workroom air levels of 2,4-DNP, determined subsequent to the death, were “normally” ≥40 mg/m³. This may underestimate breathing-zone levels, and significant dermal absorption was thought to have occurred following deposition of the chemical on the skin. Hence, no conclusions regarding fraction absorbed can be drawn from this study, but it does provide additional evidence of absorption from the inhalation route.” Dermal exposure “The 1917 and 1918 records from two French munitions factories show that the percentage of positive Derrien tests (for the metabolite 2-amino-4-nitrophenol) tended to rise during the warm months of the year (Perkins 1919). One of these factories also provided records of the percentage of clinical cases, which, although much lower than the percentage of positive Derriens, roughly paralleled the Derrien results. The increases in percentages of positive Derrien tests and clinical cases during the warmer months provide support for the idea that dermal absorption may be an important route of entry to the body, since greater exposure of the skin would be expected during the warmer months. Alternatively, the higher ambient temperatures may have caused greater loss of body water through sweating, resulting in more concentrated urine, which could result in a greater percentage of positive Derrien tests. The higher ambient temperatures would also tend to exacerbate the effects of 2,4- DNP.” “Similarly, the details of two fatal cases of 2,4-DNP in the U.S. chemical industry suggest that dermal absorption may have been a contributing factor. Two workers exposed to mists and dust of 2,4- DNP for a few months developed signs of toxicity; following treatment and rest, and then a return 17 CLH REPORT FOR 2,4-DINITROPHENOL to the job during warmer weather, they collapsed and died (Gisclard and Woodward 1946). The warmer weather during the second period of exposure (duration not specified) was thought to be a contributing factor because of the greater skin exposure and potential for increased dermal absorption; it may also have exacerbated the effects by contributing to hyperpyrexia and increased pulse produced by 2,4-DNP.”

Metabolism Quotation from ATSDR (1995 and 2011): “Some information regarding metabolism of 2,4-DNP in humans is available from cases of occupational 2,4-DNP poisoning and from a case involving ingestion of the chemical as a diet pill. The limitations of these studies include the relative lack of specificity of the methods employed to detect or quantify 2,4-DNP and its metabolites. Examination of the blood and organs of workmen who died from exposure to 2,4-DNP in the French munitions industry revealed, the presence of 2,4-DNP and its reduced metabolites (not further specified) (Perkins 1919). The compounds found in urine were 2,4-DNP, 2-amino-4-nitrophenol, 4-amino-2-nitrophenol, 2,4-diaminophenol, and other uni- dentified nitrogen compounds, which may have been glucuronide conjugation products (NRC 1982). In cases of serious 2,4-DNP poisoning, the presence of large amounts of 2-amino-4- nitrophenol in the urine were found and were the basis of a-particular test called the Derrien test, used as an indicator of exposure to 2,4-DNP. This test is a color reaction for aminonitrophenols, with the formation of a characteristic red wine to violet color for 2-amino-4-nitrophenol and a more or less yellow-orange color for 4-amino2-nitrophenol constituting a positive test. Quantitation ap- parently was limited to visual inspection for color intensity. The test is not highly specific, as pic- ramic acid also yields a red wine to violet color. The test cannot detect diaminophenols because the ability to produce the colored reaction depends on the NO2 group in aminonitrophenols. Mild cases of intoxication produced positive Derrien tests; when the urine gave a positive Derrien that increased in intensity day by day or remained fairly high, acute intoxication frequently developed. The percentage of positive Derriens did not appear to correlate with race (“white, yellow, black”) (Perkins 1919).” “In a case of fatal occupational 2,4-DNP poisoning from exposure to mists and airborne dust of 2,4-DNP in the U.S. chemical industry, the urine contained 2.08 g/L 2,4-DNP and 50 mg/L of 2- amino-4-nitrophenol (Gisclard and Woodward 1946). “ “A woman who ingested sodium 2,4-DNP at 3.5 mg/kg/day 2,4-DNP for 20 days tested positive for the presence of 2-amino-4-nitrophenol (Derrien test) and 2,4-DNP (“indicator test” not further described) in the urine (Davidson and Shapiro 1934).”

Distribution Quotation from ATSDR (1995 and 2011): Oral exposure “No studies were located regarding distribution in humans after oral exposure to 2,4-DNP.”

Inhalation exposure and dermal exposure “Examination of the blood and unspecified organs of workmen who died from exposure to 2,4-DNP in the munitions industry in France revealed the presence of 2,4-DNP and its metabolites (Perkins 1919), indicating distribution to the tissues. Analysis of the organs (not specified) of two workmen 18 CLH REPORT FOR 2,4-DINITROPHENOL who died following exposure to 2,4-DNP in the U.S. chemical industry, however, did not demonstrate the presence of the chemical or metabolites, although 2,4-DNP and 2-amino-4-nitrophenol were detected in the urine of one worker (urine was not tested in the other case) (Gisclard and Woodward 1946). Because one report (Perkins 1919) did not provide details of extraction and analytical methods, reasons for the discrepancy in results cannot be determined. In both studies, exposure may have occurred by the dermal as well as the inhalation routes.”

Elimination Quotation from ATSDR (1995 and 2011): Oral exposure “Both 2,4-DNP and its metabolite, 2-amino-4-nitrophenol, were detected in the urine of a woman who had taken the sodium salt of 2,4-DNP at 3.5 mg/kg/day 2,4-DNP for 20 days and was admitted to the hospital 5 days after cessation of DNP treatment because of severe illness (agranu- locytosis) (Davidson and Shapiro 1934). Detection of parent compound (method not described) and 2-amino-4-nitrophenol (Derrien test) occurred on the second day of hospitalization and of parent compound on the third. The bromsulphalein test for liver function showed evidence of impaired function, which may have accounted for the persistence of 2,4-DNP and 2-amino-4-nitrophenol in the body 7-8 days after cessation of intake.” Inhalation exposure and dermal exposure “In humans exposed to 2,4-DNP by inhalation, both the parent compound and metabolites appear to be excreted in the urine. 2,4-DNP and its metabolites have been detected in the urine of workmen who died from exposure to 2,4-DNP in the munitions industry in France; quantitative exposure or urinary data were not provided (Perkins 1919). In addition, a yellow staining of the skin was observed after workers perspired profusely, indicating that 2,4-DNP was excreted in the sweat. As discussed in Section 2.3.1.1, a metabolite of 2,4-DNP, 2-amino-6nitropheno1, was commonly detected in the urine of such workmen; quantitative data were not provided (Perkins 1919). In a case of fatal occupational 2,4-DNP poisoning in the U.S., the urine contained 2.08 g/L of 2,4-DNP and 50 mg/L of 2-amino-4-nitrophenol (Gisclard and Woodward 1946). In both occupational studies, exposure may have occurred by the dermal as well as inhalation routes.”

4.1.3 Summary and discussion on toxicokinetics Quotation from the ATSDR (1995 and 2011): “The toxicokinetics of 2,4-DNP in humans and animals have not been studied systematical- ly. The available data from human case reports and experimental animal studies indicate that 2,4- DNP is readily absorbed by the oral and inhalation routes, and possibly by the dermal route. Some evidence about distribution is available suggesting that a portion of the 2,4-DNP in the blood is bound to serum proteins and that the unbound fraction enters organs such as the eye. 2,4-DNP is rapidly metabolized via reduction of the nitro groups; the parent compound and metabolites are excreted in the urine.”

19 CLH REPORT FOR 2,4-DINITROPHENOL

4.2 Acute toxicity

4.2.1 Non-human information

4.2.1.1 Acute toxicity: oral Table 10: Summary table of relevant acute oral toxicity studies Method Results Remarks Reference Rat No mortality occurred at doses 2 (reliable with Spencer et al. oral (gavage) in olive oil contain- of 10 – 27 mg/kg bw restrictions) (1948) ing 5 -10 % gum Arabic solution. 37 % (11/30) mortality occurred Test material: 2,4- white rats, young mature, both at 30 mg/kg bw dinitrophenol sexes (9 to 40 animals per dose 90 % (18/20) mortality occurred group). at 40 mg/kg bw Following doses were applied: 10, 100 % (20/20) mortality oc- 20, 23, 25, 27, 30, 40, 50, 60, 70, curred at 100 mg/kg bw 80 and 100 mg/kg b.w.

Rat LD50 71 mg/kg bw (95 %) con- 4 (not assignable) Kaiser, 1964 Oral findence limits: 57-89 Test material: 2,4- Weanling male rats (Sherman- dinitrophenol strain) no information on doses and numbers of animals

Rat LD50: 30 mg/kg b.w. 4 (not assignable) Schafer (1972) Oral: no further details Test material: 2,4- dinitrophenol

Rat LD0: 20 mg/kg bw 4 (not assignable) Dow chemicals (1940) cited ac- Oral: gavage LD100: 60 mg/kg bw Test material: 2,4- cording to White rats, no further details dinitrophenol ATSDR (1995)

Rat LD50: 30 mg/kg bw 4 (not assignable) Dow chemicals Oral: gavage Test material: 2,4- (1950) cited according to White rats, no further details dinitrophenol ATSDR (1995)

Rat LD50: 30 mg/kg bw 4 (not assignable) According to Oral: no further details Test material: 2,4- HSDB database dinitrophenol

Mouse LD50 72 mg/kg b.w. (95 % con- 4 (not assignable) Kaiser, 1964 Oral findence limits: 62-84 mg/kg Test material: 2,4- b.w.) Weanling male mice (white C.F.1- dinitrophenol strain) no information on doses and numbers of animals

Mouse LD50: 45 mg/kg bw 4 (not assignable) according to Oral: No further details Test material: 2,4- RTECS-Website dinitrophenol

Rabbit LD50: 30 mg/kg bw 4 (not assignable) according to Oral: No further details Test material: 2,4- RTECS-Website dinitrophenol

Rabbit LD50: 30 mg/kg bw 4 (not assignable) according to Oral: No further details Test material: 2,4- HSDB database dinitrophenol

20 CLH REPORT FOR 2,4-DINITROPHENOL

Method Results Remarks Reference

Guinea pig LD50: 81 mg/kg bw 4 (not assignable) according to Oral: no further details Test material: 2,4- RTECS-Website dinitrophenol

Dog LDLo: 30 mg/kg bw 4 (not assignable) according to Oral: No further details Test material: 2,4- RTECS-Website dinitrophenol

Dog LD50: 20 -30 mg/kg bw 4 (not assignable) according to Oral: No further details Test material: 2,4- RTECS-Website dinitrophenol

Cat LD50: 75 mg/kg bw 4 (not assignable) according to Oral: No further details Test material: 2,4- RTECS-Website dinitrophenol

Reference: Spencer et al., 1948 Study design: The oral toxicity of 2,4-dinitrophenol was investigated in young mature white rats of both sexes (no further information on strain and age of the animals was given, furthermore no details were given on the distribution of male and female rats). Dinitrophenol was applied in olive oil containing a 5 – 10 % gum Arabic solution via gavage. The volume of oil given to each rat was less than 3 ml and was usually of the order of 1 ml. Results: All of the rats that survived were observed until it was certain that they had fully recovered (usually about two weeks). Deaths that resulted from the treatment with dinitrophenol were attributed to be due mainly to their pyretic effect; as a rule, death occurred an hour or two after the treatment or not at all. A total number of 239 animals were treated with dinitrophenol. The results are presented in table 12. It was concluded, that dinitrophenol was a rapidly acting agent. A survival dose of 27 mg/kg bw and a lethal dose of 100 mg/kg bw were defined. Table 11: Acute oral toxicity of dinitrophenol (Spencer et al., 1948) Dose [in mg/kg bw] No of animals treated No died Percentage 10 9 0 0 20 20 0 0 23 10 0 0 25 10 0 0 27 10 0 0 30 30 11 37 40 20 18 90 50 20 17 85 60 20 19 95 70 30 15 50 80 40 18 45 100 20 20 100 21 CLH REPORT FOR 2,4-DINITROPHENOL

Conclusion:

It can be concluded that dinitrophenol has a very steep dose-response and that the LD50 can be estimated for a dose between 30 and 40 mg/kg bw. The study has been performed before the establishment of guidelines and good laboratory practice. Some information is lacking such as the precise age of the animals and the ratio of male and female rats in each dose group. However, these information gaps are considered as minor important and the total conclusion is Klimisch score 2 (reliable with restrictions).

Reference: Kaiser, 1964 Study design: The oral toxicity of 2,4-dinitrophenol was investigated in weanling male rats of the Sherman strain weighing 40 – 55 g when received. Male weanling C.F. 1 white mice weighing 15-18 g were also used. The animals were deprived of solid food for approximately 18 hours prior to dosing. Solid food was again given to the animals 30 minutes after dosing and was available for the duration of the period of observation, which lasted at least 7 days after treatment. A suspension of dinitrophenol was prepared using a 1 % aqueous starch solution. Results: The following signs of toxicity were observed in rats and mice: tremors, prostration, increase in respiratory rate, tonic convulsions and rigidity of limbs just prior to or immediately after signs of death. The survivors experienced a temporary increase in respiration. Their weight gain was similar to that of the controls.

LD50 values of 71 mg/kg bw were presented for rats and for mice the LD50 values were presented with 72 mg/kg bw. Conclusion:

LD50 values were almost identical in rats and mice. The study has been performed before the establishment of guidelines and good laboratory practice. However, important information is lacking such as the number of animals investigated and doses of dinitrophenol applied. Due to these important deficiencies the conclusion is that the Klimisch score is 4 (not assignable). Nevertheless, the LD50 values will be used as supporting information.

Further publications and information mentioned in Table 11 The publication of Schafer (1972) summarizes the acute oral toxicity of 369 compounds in different species. Table 1 of this publication referred to a LD50 of 30 mg/kg bw in rats without any further information. A study report from Dow Chemicals (1940) was cited by the ATSDR (1995) with a 100 % survival at the dose of 20 mg/kg bw and a 100 % lethal dose of 60 mg/kg bw in white rats treated once by gavage. No further information was available.

Another study report from Dow Chemicals (1950) was cited by the ATSDR (1995) with a LD50 value of 30 mg/ kg bw for white rats treated once by gavage. No further information was available.

22 CLH REPORT FOR 2,4-DINITROPHENOL

It is possible, that the Schafer review (1972) referred to this study report since it mentioned an identical LD50 value. However, without any further information it cannot be investigated any further.

On the RTECS website information was available for five species: mouse (LD50: 45 mg/kg bw), rabbit (LD50: 30 mg/kg bw), Guinea pig (LD50: 81 mg/kg bw), dog (LDLo: 30 mg/kg bw) and cat (LD50: 75 mg/kg bw). No further information was accessible.

In the HSDB database information was available for three species: rat (LD50: 30 mg/kg bw), rabbit (LD50: 30 mg/kg bw) and dog (LD50: 20 - 30 mg/kg bw). No further information was accessible. Due to significant deficiencies in the description these publications and information were allocated a Klimisch score of 4 (not assignable). However, it will be used as supporting information.

4.2.1.2 Acute toxicity: inhalation No data were available

4.2.1.3 Acute toxicity: dermal Table 12: Summary table of relevant acute dermal toxicity studies Method Results Remarks Reference Guinea pig No mortality occurred at doses 2 (reliable with Spencer et al. Dermal, alcoholic solution of 2,4- of 100 and 200 mg/kg bw restrictions) (1948) dinitrophenol was applied to the 20 % (1/5) mortality occurred at Test material: 2,4- clipped abdomen. 300 and 400 mg/kg bw dinitrophenol Guinea pig, both sexes (5 animals 40 % (2/5) mortality occurred at per dose group). 500 mg/kg bw Following doses were applied: 100 % (5/55) mortality occurred 100, 200, 300, 400, 500, 700 and at 700 and 1000 mg/kg bw 1000 mg/kg bw

Reference: Spencer et al., 1948 Study design: Guinea pigs of both sexes (heterogenous stock from a commercial supplier) were treated with a single dose of dinitrophenol in alcoholic solution on the clipped abdomen. Each guinea pig was restrained on an animal board in such a manner that the treated area could be kept wet with ethanol during the four-hour period following the application of the test material in order to facilitate its absorption. At the end of this period, the surviving animals were removed from the boards, band- aged so as to prevent oral ingestion, caged, and observed until it was certain that they fully recovered. Results: A total number of 35 animals were treated dermally with dinitrophenol. No mortality occurred at 100 and 200 mg/kg bw. 20 % (1/5) mortality occurred at 300 and 400 mg/kg bw, at 500 mg/kg bw the mortality increased to 40 % (2/5). A mortality of 100 % occurred at 700 and 1000 mg/kg bw. A survival dose of 200 mg/kg bw and a lethal dose of 700 mg/kg bw were defined. Conclusion:

It can be concluded that dinitrophenol has a steep dose-response and that the LD50 can be estimated for a dose between 500 and 700 mg/kg bw. The study has been performed before the establishment

23 CLH REPORT FOR 2,4-DINITROPHENOL of guide lines and good laboratory practice. Some information is lacking such as the precise age of the animals and the ratio of male and female guinea pigs in each dose group. However, these information gaps are considered as minor important and the total conclusion is Klimisch score 2 (reliable with restrictions).

4.2.1.4 Acute toxicity: other routes No data were available.

4.2.2 Human information Dinitrophenol has been used historically in dyes, wool preservatives, herbicides, and explosives since the early 20th century. In the 1930s it has been also used as an agent to lose weight (Yen & Ewald, 2012). Several cases of dinitrophenol poisonings have been published in the 1930s. The evaluation of the ATSDR (1995) is quoted as follows: “Little information is available regarding death in humans after acute oral exposure to 2,4- DNP. A case report details the death of an 80-kg man who took ≈ 46 mg 2,4-DNP/kg as the sodium salt, followed by another 46 mg/kg dose 1 week later (Tainter and Wood 1934). The first dose produced a high fever; the second dose resulted in admission to the hospital 6.5 hours later because of hyperpnea and chest pain. The rectal temperature was 105 °F, and pulse was rapid (as high as 146 beats per minute). Despite the administration of aspirin, the temperature rose to 105.7 °F by 10.5 hours following ingestion of the drug. Death occurred 0.5 hours later, with rigor mortis setting in 10 minutes after death and the temperature rising to ≈115 °F by 20 minutes after death. The clinical signs and the autopsy and histological findings were considered by the authors to be similar to those seen in heat stroke. A woman who took 7 mg/kg/day 2,4-DNP as the sodium salt for 5 days was admitted to the hospital in a comatose condition and subsequently died (Poole and Haining 1934). She had complained of headache, backache, weakness, dizziness, shortness of breath, and excessive perspiration. Her temperature was at least 101.8 °F, pulse 140 beats per minute, and respiratory rate 56 per minute. Upon autopsy and histological examination, hyperemic and hemorrhagic lungs, degeneration of renal tubules and liver cells, segmentation and fragmentation of cardiac muscles, and hemorrhagic spleen, stomach mucosa, spinal cord, pons, and medulla were found. Slight ganglion cell degeneration was found in the pons. In another case, a psychiatric patient was given sodium 2,4-DNP in an experimental study to determine whether 2,4-DNP would be beneficial in treating depression (Masserman and Goldsmith 1934). Over the course of 14 days, she had been given 2.66 mg/kg/day 2,4-DNP. She died after her pulse increased to 148 beats per minute and respirations to 48 per minute, her temperature rose to 102 °F, she became comatose, and blood pressure fell to 36/0. Because autopsy was delayed for 4 days, no conclusions regarding histopathological lesions could be made. There were no deaths, however, in a number of clinical and experimental studies in which obese or normal subjects were given 2,4-DNP or its sodium salt at oral dosages of 1.2-4.3 mg/kg/day 2,4-DNP for ≤14 days (Castor and Beierwaltes 1956; Cutting et al. 1934; Cutting and Tainter 1933; MacBryde and Taussig 1935; Stockton and Cutting 1934; Tainter et al. 1935b). “

“In studies of intermediate-duration oral exposure to 2,4-DNP, cases of death from agranu- locytosis have been attributed to 2,4-DNP. These cases occurred during the usual dosing regimens for weight loss, employing increasing doses in one case from 2.9 to 4.3 mg/kg/day of 2,4-DNP for 6 weeks (Dameshek and Gargill 1934); a dose of 1.03 mg/kg/day 2,4-DNP for 46 days in another case (Goldman and Haber 1936); and in another, from 0.62 to 3.8 mg/kg/day 2,4-DNP as sodium 2,4- DNP for 41 days (Silver 1934). In all cases, the patients were under medical supervision. Several clinical studies regarding the effects of 2,4-DNP or its sodium salt in obese and non-obese humans taking the drug for an intermediate duration at doses of 3.5-5.27 mg/kg/day 2,4-DNP have reported

24 CLH REPORT FOR 2,4-DINITROPHENOL no deaths from this treatment (Cutting et al. 1934; Grant and Schube 1934; Looney and Hoskins 1934; MacBryde and Taussig 1935; Simkins 1937a, 1937b; Tainter et al. 1934a, 1935b). A woman who took 3-5 tablets a day of 2,4-DNP for several months, discontinued its use for 3 months, and then resumed taking 5 tablets a day for 1 week, became ill only after resumption of dosing and subsequently died (Lattimore 1934). The data reported were insufficient to determine a dose in this case. It is not known why this woman tolerated the treatment for several months without developing any signs of illness, then subsequently became ill and died within 1 week after resumption of the same dose.”

More recent reports were described in the following part. McFee et al., (2004) reported the case of a 22-year-old man with a change in mental status 16 hours after his last dose of dinitrophenol. The patient admitted he had taken 600 mg dinitrophenol each day for the last four days. The patient was obese and showed a body temperature of 38.9 oC. The patient received mechanical cooling and i.v. treatment with midazolam. He experienced progressive bradycardia, the cardiac status deteriorated; he developed asystole and finally died. Calculating with the last of four doses of 600 mg each and assuming a body weight of 97 kg (corresponding to a body height of 1.8 m and a body-mass-index of 30), the dinitrophenol dose would be 6.2 mg/kg bw. Suozi et al. (2005) reported the case of a 24-year-old man who collapsed during workout. Paramed- ics received a bottle of capsules filled with yellow powder and a page titled “DNP and weight loss”. In the hospital the patients’ temperature rose to 40.8 oC despite cooling measures and rigorous fluid hydration. The patient developed pulmonary edema, suffered multiple organ damage, including renal failure, went into cardia arrest and subsequently died. Tewari et al. (2009) reported the case of 27-year-old woman with a BMI of 33, complaining of fa- tigue, nausea and excessive sweating. She admitted to starting a new diet tablet (bought over the internet) a week before her admission. She had doubled the recommended doses for faster results. Heart rate was 140 bpm, blood pressure 122/86 and the temperature 38 oC. The patient received saline and diazepam. Seven hours after admission she desaturated to < 90 % and became asystolic. Despite resuscitation measure the patient died. Bartlett et al. (2010) published a case report on a 46-year-old white man who ingested 14 capsules containing 200 mg dinitrophenol each (total dose 2.8 g) with suicidal intent. Fourteen hours after ingestion he was brought to the emergency department. The patient was tachycardic (170 bpm), normotensive (140/80 mm Hg), sweaty, flushed and agitated but oriented. The body temperature was 37.8 oC. Supportive management included fluid resuscitation, active cooling measures (including paracetamol and dantrolene) and diazepam for agitation. However, the situation continued to deteriorate with worsening hypotension and the patient lost cardiac output. He died 7 h after admission and 21 h after ingestion of 2.8 g Dinitrophenol (Bartlett et al., 2010). Assuming a body weight of 70 kg, the dinitrophenol dose would be 40 mg/kg bw. Siegmueller & Narasimhaiah (2010) published a case report of a man who ingested 14 tablets containing 200 mg dinitrophenol each (total dose 2.8 g) with suicidal intent. Twelve hours after ingestion he was brought to the emergency department. On admission the patient was conscious, complaining of dizziness, back and abdominal pain, and reporting frequent diarrhoea as well as vomiting. He was sweating profusely and appeared clinically dehydrated. His temperature was 38.4 oC, blood pressure 104/64 mm Hg and a heart rate of 150/min. The immediate management consisted of intravenous fluid resuscitation and treatment of the hyperkalaemia with calcium gluconate and an insulin/dextrose infusion. The condition of the patient deteriorated and body temperature rose to 39.5 oC. Following intubation dantrolene was administered; however, the patient suffered an asystolic cardiac arrest and died three hours after admission (Siegmueller & Narasimhaiah 2010). As- suming a body weight of 70 kg, the dinitrophenol dose would be 40 mg/kg bw. 25 CLH REPORT FOR 2,4-DINITROPHENOL

Kamour et al. (2015) reviewed the inquiries to the National Poisons Information Service in the United Kingdom from 2007 to 2013. Five fatal poisonings were reported. For three of the five cases data on the ingested dose were presented (2800, 3200 mg and 2980 mg, respectively). All three casualties were male and assuming a body weight of 70 kg, the dinitrophenol doses would be 40, 46 and 43 mg/kg bw, respectively (see Table 13). No information was available whether the intake of dinitrophenol happened with suicidal intent. In Germany, the Federal Institute for Risk Assessment (BfR) collects information on poisoning in- cidents in a poison information database. In 2015 an evaluation has been performed for fatal poisonings by dinitrophenol (BfR, 2015) (see Table 13 and Table 14). In two cases information was available on the dinitrophenol dose (4000 mg each). The casualties were male and assuming a body weight of 70 kg, the dinitrophenol dose would be 57 mg/kg b.w. (see Table 13). Table 13: Fatal oral poisonings with information about doses applied Reference Dose [mg] Body weight Dose in mg/kg bw Number of doses [N] ATSDR (1995) 3680 80 kg male patient, intake of two 46 mg/kg bw 2 doses within one week ATSDR (1995) - A woman took DNP for five dayss. 7 mg/kg bw 5 No information was available on bw. However a dose per kg bw was presented. ATSDR (1995) - A female patient received DNP for 2.66 mg/kg bw 14 14 days. No information was available on bw. However a dose per kg bw was presented. ATSDR (1995) - A patient received DNP for 6 weeks. 2.9 to 4.3 mg/kg bw 42 No information was available on bw. However a dose per kg bw was presented. The dose was increased from 2.9 to 4.3 mg/kg bw/day ATSDR (1995) - A patient received DNP for 46 days. 1.03 mg/kg bw 46 No information was available on bw. However a dose per kg bw was presented. ATSDR (1995) - A patient received DNP for 6 weeks. 0.62 to 3.8 mg/kg bw 42 No information was available on bw. However a dose per kg bw was presented. The dose was increased from 0.62 to 3.8 mg/kg bw/day McFee et al. (2004) 600 The male patient was obese. Estima- 600 mg / 97 kg = 6.2 mg/kg 4 tion of bw with 97 kg for 1.80 m bw height and body mass index of 30 (corresponding to obesity). The patient took DNP for four days. Bartlett et al. (2010) 2800 mg with sui- Male, bw was estimated with 70 kg 2800 mg / 70 kg = cidal intent 40 mg/kg bw 1 Siegmueller & Nara- 2800 mg with sui- Male, bw was estimated with 70 kg 2800 mg / 70 kg = simhaiah (2010) cidal intent 40 mg/kg bw 1

26 CLH REPORT FOR 2,4-DINITROPHENOL

Reference Dose [mg] Body weight Dose in mg/kg bw Number of doses [N] Kamour et al. (2015) 2800 mg Male, bw was estimated with 70 kg 2800 mg / 70 kg = 1 40 mg/kg bw Kamour et al. (2015) 2980 mg Male, bw was estimated with 70 kg 2980 mg / 70 kg = 1 43 mg/kg bw Kamour et al. (2015) 3200 mg Male, bw was estimated with 70 kg 3200 mg / 70 kg = 1 46 mg/kg bw BfR (2015) 4000 mg Male, bw was estimated with 70 kg 4000 mg / 70 kg = 1 57 mg/kg bw BfR (2015) 4000 mg with sui- Male, bw was estimated with 70 kg 4000 mg / 70 kg = cidal intent 57 mg/kg bw 1

Additionally, fatal poisonings without information on the dinitrophenol doses were compiled in Table 14 to demonstrate the multitude of fatalities. Table 14: Fatal oral poisonings without information about doses applied Reference Basic information More information ATSDR (1995) female, intake over several month No further information Suozzi et al. (2005) male Member of a health club Miranda et al. (2006) female DNP was used for weight-loss Miranda et al. (2006) male Use of bodybuilding supplements Tewari et al. (2009) female, BMI of 33 DNP was used to support a diet Grundlingh et al. (2011) female Suicidal intent Grundlingh et al. (2011) male Use of bodybuilding supplements BfR, (2015) female No further information Kamour et al. 2015 male No further information Kamour et al. 2015 female No further information

Jiukun et al. (2010) reported two fatal cases of dinitrophenol poisoning caused by non-oral exposure. A 49-year-old woman and 41-year-old man were poisoned when they were recycling useless nylon bags that were used as packages of dinitrophenol. They wore no protective clothing and only wore ordinary masks after seeing a lot yellow powder. According to statements given by the victims and witnesses, oral exposure can be excluded. The skin of hands, feet, cnemis and forearms were heavily stained by dinitrophenol.

4.2.3 Summary and discussion of acute toxicity There is long history of oral poisonings by dinitrophenol in humans. The ATSDR (1995) compiled several cases from the 1930s with doses in the range of less than 1 mg dinitrophenol/kg bw and 7 mg/kg bw leading to a fatal outcome. During recent years new case reports have been published with estimated doses of 6.2 mg/kg bw in one case and seven cases in a dose range of 40 to 57 mg/kg bw. It should be noted that three of the seven high-dose cases rely on suicidal intent. Fur-

27 CLH REPORT FOR 2,4-DINITROPHENOL thermore, ten additional fatal poisonings (Table 14) demonstrate the serious problem in the acute toxicity of dinitrophenol. Only very limited information is available on non-oral poisonings. However, the case report from China clearly shows that a dermal exposure towards dinitrophenol can lead to a fatal outcome. For acute oral toxicity in animals, only one study can be considered for the evaluation. The rat study by Spencer et al. (1948) came close to fulfil the OECD TG requirements, although both sexes have been used in the study. All other reports indicated the LD50 values and the test species, but showed serious deficiencies, mostly all experimental details were lacking. The Spencer study yielded a range from 30 to 40 mg/kg bw for the LD50 value. Three reports referenced to a LD50 of 30 mg/kg bw in rats. It is not possible to investigate, if these values rely on the same study. Another LD50 value in rats was calculated with 71 mg/kg bw. Finally, a study from Dow Chemicals was cited in the ATSDR with a LD0 of 20 mg/kg bw and a LD100 of 60 mg/kg bw, therefor the LD50 is somewhere between 20 and 60 mg/kg bw. Despite the deficiencies of most of the studies and reports presented here, the range of LD50 in rats is in the range between 30 and 71 mg/kg bw.

Additional information on oral toxicity in other animals resulted in two studies in mice with LD50 of 45 and 72 mg/kg bw, two study in rabbits with 30 mg/kg bw, two studies in dogs with a LD50 of 20 – 30 mg/kg bw and a LDLo of 30 mg/kg bw, one study in cats with a LD50 of 75 mg/kg bw and one study in Guinea pigs with 81 mg/kg bw. Since all studies have serious deficiencies they can only be used to confirm the range of acute oral toxicity of dinitrophenol (30 to 81 mg/kg bw).

Finally, only one acute dermal toxicity study was available (Spencer et al., 1948), showing a LD50 in the range of 500 to 700 mg/kg bw. As in the oral toxicity study, the use of both sexes was the major deficiency in the dermal toxicity study.

4.2.4 Comparison with criteria oral

Spencer et al. (1948) is selected as key study. This study gave a range for acute oral LD50 in the rat from 30 to 40 mg/kg bw. An acute oral LD50 of 30 to 40 mg/kg bw fulfils the requirement for classification in Category 2 (LD50 ranging between 5 and 50 mg/kg bw). For the calculation of the ATE the average of the doses below and above the LD50 have been used and yielded a value of 35 mg/kg bw.

Furthermore, the human evidence will be considered: Twelve of 14 fatal poisonings with information on the dose taken showed doses < 50 mg/kg bw. Six of the 14 fatal poisonings presented with doses < 10 mg/kg bw. However, these victims repeatedly ingested doses of DNP as presented in Table 13. Ten additional fatal poisonings without detailed information on the intake highlight the relevance of the acute toxicity of DNP for humans. dermal

Spencer et al. (1948) is selected as key study. This study gave a range for acute dermal LD50 in Guinea pigs from 500 to 700 mg/kg bw. An acute dermal LD50 of 500 to 700 mg fulfils the requirement for classification in Category 3 (LD50 ranging between 200 and 1000 mg/kg bw). For the calculation of the ATE the average of the doses below and above the LD50 have been used and yielded a value of 600 mg/kg bw.

28 CLH REPORT FOR 2,4-DINITROPHENOL

4.2.5 Conclusions on classification and labelling According to Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging, dinitrophenol should be classified as Acute Tox. 2, H300, thereby replacing the current classification of Acute Tox. 3, H301. It is proposed to assign an ATE of 35 mg/kg bw for acute oral toxicity. The available data do not warrant a change in the current classification for acute dermal toxicity (Acute Tox. 3, H311). It is proposed to assign an ATE of 600 mg/kg bw for acute dermal toxicity. No data have been obtained for acute inhalation toxicity. Therefor no change is proposed in the current classification for acute inhalative toxicity (Acute Tox. 3, H331).

4.7 Repeated dose toxicity Table 15: Summary table of relevant repeated dose toxicity studies Method Results Remarks Reference Subacute oral toxicity testing on In the highest dose group 6 of 12 1 (reliable without Koizumi et al. rats (according to test guideline of females treated died and 2 of 12 restrictions) (2001) the Japanese Chemical Control males treated died. Test material: 2,4- Act). Test procedure is similar to All animals in high dose group dinitrophenol OECD TG 407. showed repeatedly symptoms of toxicity such as decrease in Sprague-Dawley SPF rats locomotor activity, prone posi- [Crj:DC(SD)IGS] were treated tion, ptosis, panting, crawling orally by gastric intubation once a position and salivation. day for 28 days. Doses were 0, 3, 10, 30, 80 mg dinitrophenol/kg bw/day. Six animals per dose and sex were treated except for the 30 and 80 mg/kg b.w./day groups, where 12 animals per sex were used for a recovery protocol.

2,4-dinitrophenol (purity 85.2 %) was suspended in 1 w/v% methyl- cellulose solution.

4.7.1 Non-human information

4.7.1.1 Repeated dose toxicity: oral A subacute oral toxicity study was performed on young Sprague-Dawley rats, aged 5 to 6 weeks (Koizumi et al., 2001). 2,4-dinitrophenol (purity 85.2 %) was suspended in a methycellulose solution and administered per gavage. Rats were treated with doses of 0, 3, 10, 30, 80 mg/kg bw/day for 28 days. Recovery groups (0, 30, 80 mg/kg bw/day) were maintained for 2 weeks without treatment and fully examined at 11 to 12 weeks of age. The number of animals for each sex/dose was 6 for both scheduled-sacrifice and recovery. Rats were examined for general behaviour, body weight, food consumption, urinalysis, hematology and blood biochemistry, necropsy finding, organ weights and histopathology finding in compliance the Test guideline of the Japanese Chemical Control Act (Koizumi et al., 2001). There was no mortality in the control group and in the lower dose groups (3, 10, 30 mg/kg bw/day). Symptoms of toxicity such as decrease in locomotor activity and salivation were observed in the 30

29 CLH REPORT FOR 2,4-DINITROPHENOL mg/kg bw/day-group mostly after the first dosing only. No changes in histopathology or organ weights were observed at 30 mg/kg bw/day or lower (Koizumi et al., 2001). In the highest dose group 6 of 12 females and 2 of 12 males died. All animals in the high dose group showed repeatedly symptoms of toxicity such as decrease in locomotor activity, prone position, ptosis, panting, crawling position, salivation. Tonic convulsion was observed in 2 of 12 males and 4 of 12 females. Rigidity was observed in 2 of 12 males and 5 of 12 females. The relative liver weights were increased in both sexes of the scheduled-sacrifice group and this persisted through the recovery period. Relative organ weights for brain, kidneys and testes were increased only in males. On histopathological examination, mineralization of the corticomedullary junction in kidneys was observed in both sexes in the scheduled-sacrifice and recovery group, but the change was only sta- tistically significant in males of the scheduled-sacrifice group. On haematological examination, increase in haemoglobin and haematocrit during the treatment, and decrease in red blood cell count, haemoglobin and haematocrit in the recovery group were observed, limited to males. (Koizumi et al., 2001). It can be concluded that dinitrophenol shows a clear toxicity at 80 mg/kg bw/day with a mortality of 6 of 12 females and 2 of 12 males. In comparison to this clear toxicity the other effects on organ weight and on histopathology are irrelevant. The study has been performed according to the Japa- nese guideline and seems to be equivalent to the OECD TG 407. The study has been performed under GLP conditions and therefor the total conclusion is Klimisch score 1 (reliable without restrictions).

4.7.1.2 Repeated dose toxicity: inhalation There is no information available.

4.7.1.3 Repeated dose toxicity: dermal There is no information available.

4.7.1.4 Repeated dose toxicity: other routes There is no information available.

4.7.1.5 Human information There are several very old studies from the 1930s which are summarized in the ATSDR (1995).

4.7.1.6 Other relevant information

4.7.1.7 Summary and discussion of repeated dose toxicity Dinitrophenol clearly shows toxicity in a repeated dose toxicity study on rats. In a 28-day oral toxicity study 6 of 12 female rats and 2 of 12 male died during treatment with 80 mg/kg bw/day. The study seems to be reliable without restrictions.

4.8 Specific target organ toxicity (CLP Regulation) – repeated exposure (STOT RE) For details see section 4.7.

30 CLH REPORT FOR 2,4-DINITROPHENOL

4.8.1 Summary and discussion of repeated dose toxicity findings relevant for classification as STOT RE according to CLP Regulation There is long history of oral poisonings by dinitrophenol in humans. The ATSDR (1995) compiled several studies from the 1930s with different doses. However, these studies are not necessary to confirm the toxicological profile of dinitrophenol. The key study on the repeated dose toxicity of dinitrophenol in animals is a guideline study performed in rats. In a 28-day oral toxicity study 6 of 12 female rats and 2 of 12 male died during treatment with 80 mg/kg b.w./day. The study seems to be reliable without restrictions.

4.8.2 Comparison with criteria of repeated dose toxicity findings relevant for classification as STOT RE Kouizumi et al. (2001) was selected as key study. In a 28-day oral toxicity study on rats a dose of 80 mg/kg bw/day elicited a mortality of 6 out of 12 females and 2 out of 12 males. Mortality is the most severe endpoint in toxicity. The value of 80 mg/kg bw/day corresponds to the guidance values for category STOT RE2 (30 < C =< 300, for 28-day studies). It is also evident, that the effects are not sufficient to classify into STOT RE1 (C = < 30).

4.8.3 Conclusions on classification and labelling of repeated dose toxicity findings relevant for classification as STOT RE According to Regulation (EC) 1272/2008 on Classification, Labelling and Packaging, the classification of dinitrophenol as STOT RE2, H373 should be maintained.

31 CLH REPORT FOR 2,4-DINITROPHENOL

7 REFERENCES ATSDR (1995): Toxicological Profile for Dinitrophenols, volume 352. https://www.atsdr.cdc.gov/ToxProfiles/tp.asp?id=729&tid=132 ATSDR (2011): Addenum to the toxicological profile for Dinitrophenols, 11. https://www.atsdr.cdc.gov/toxprofiles/dintrophenols_addendum.pdf Bartlett J., Brunner M., and Gough K. (2010): Deliberate poisoning with dinitrophenol (DNP): an unlicensed weight loss pill. Emerg Med J 27 (2), 159-160. DOI: 10.1136/emj.2008.069401 BfR (2015): Nahrungsergänzungsmittel, die Dinitrophenol (DNP) enthalten, können zu schweren Vergiftungen bis hin zu Todesfällen führen. Bundesinstitut für Risikobewertung. http://www.bfr.bund.de/cm/343/nahrungsergaenzungsmittel-die-dinitrophenol-dnp-enthalten- koennen-zu-schweren-vergiftungen-bis-hin-zu-todesfaellen-fuehren.pdf) Grundlingh, J., Dargan, P. I., El-Zanfaly, M. and Wood, D. M. (2011): 2,4-dinitrophenol (DNP): a weight loss agent with significant acute toxicity and risk of death. J Med Toxicol 7(3), 205-212 Harris O.M.A. and Corcoran J.J. (1995): Toxicology profile for dinitrophenols (peer review). U. S Department of health and Human Services, Public Health Service, Agency for toxic Substances and Disease Registry (ATSDR) HSDB database: 2,4-Dinitrophenol https://toxnet.nlm.nih.gov/cgi-bin/sis/search2/f?./temp/~YXeh4v:1 Jiukun J., Zhihua Y., Weidong H. and Jiezan W. (2010): 2, 4-dinitrophenol poisoning caused by non-oral exposure. Toxicol Ind Health 27 (4), 323-327. DOI: doi:10.1177/0748233710387004 Kaiser J.A. (1964): STUDIES ON THE TOXICITY OF DISOPHENOL (2,6-DIIODO-4- NITROPHENOL) TO DOGS AND RODENTS PLUS SOME COMPARISONS WITH 2,4- DINITROPHENOL. Toxicol Appl Pharmacol 6, 232-244 Kamour A., George N., Gwynnette D., Cooper G., Lupton D., Eddleston M., Thompson J.P., Vale J.A., Thanacoody H.K., Hill S., and Thomas S.H. (2015): Increasing frequency of severe clinical toxicity after use of 2,4-dinitrophenol in the UK: a report from the National Poisons Information Service. Emerg Med J 32 (5), 383-386. DOI: 10.1136/emermed-2013-203335 Koizumi, M., Yamamoto, Y., Ito, Y., Takano, M., Enami, T., Kamata, E. and Hasegawa, R. (2001): Comparative study of toxicity of 4-nitrophenol and 2,4-dinitrophenol in newborn and young rats. J Toxicol Sci 26(5), 299-311 McFee R.B., Caraccio T.R., McGuigan M.A., Reynolds S.A. and Bellanger P. (2004): Dying to be thin: a dinitrophenol related fatality. Vet Hum Toxicol 46 (5), 251-254 Miranda, E. J., McIntyre, I. M., Parker, D. R., Gary, R. D. and Logan, B. K. (2006): Two deaths attributed to the use of 2,4-dinitrophenol. J Anal Toxicol 30 (3), 219-222 RTECS (2000): Phenol, 2,4 - dinitro - http://webapp1.dlib.indiana.edu/virtual_disk_library/index.cgi/5678550/FID2757/nioshdbs/rtecs/sl2 ab980.htm Schafer E.W. (1972): The acute oral toxicity of 369 pesticidal, pharmaceutical and other chemicals to wild birds. Toxicol Appl Pharmacol 21 (3), 315-330. DOI: http://dx.doi.org/10.1016/0041- 008X(72)90151-2

32 CLH REPORT FOR 2,4-DINITROPHENOL

Siegmueller C. and Narasimhaiah R. (2010): 'Fatal 2,4-dinitrophenol poisoning... coming to a hospital near you'. Emerg Med J 27 (8), 639-640. DOI: 10.1136/emj.2009.072892 Spencer H.C., Rowe V.K., and et al. (1948): Toxicological studies on laboratory animals of certain alkyldinitrophenols used in agriculture. J Ind Hyg Toxicol 30 (1), 10-25

Suozzi J.C., Rancont C.M., and McFee R.B. (2005): DNP 2,4-dinitrophenol: a deadly way to lose weight. Jems 30 (1), 82-89; quiz 90-81

Tewari A., Ali T., O'Donnell J., and Butt M.S. (2009): Weight loss and 2,4-dinitrophenol poisoning. Br J Anaesth 102 (4), 566-567. DOI: 10.1093/bja/aep033

Yen M. and Ewald M.B. (2012): Toxicity of weight loss agents. J Med Toxicol 8 (2), 145-152. DOI: 10.1007/s13181-012-0213-7